Phosphate is an essential nutrient that contributes to all aspects of cellular physiology. Despite its essentiality, phosphate can also be toxic, thus regulation of phosphate uptake and homeostasis is critical for all forms of life. This is exemplified by the observation that disrupting these processes reduces the virulence of many enterobacterial pathogens. Staphylococcus aureus is a devastating pathogen that is a serious threat to human health due to the continued emergence of antibiotic resistance. Surprisingly, neither phosphate uptake nor homeostasis have been systematically studied in S. aureus. This is despite the fact that phosphate homeostasis in S. aureus is linked to changes in daptomycin resistance. Phosphate homeostasis has been most comprehensively studied in Escherichia coli where it is controlled by a two-component system known as PhoBR (PhoPR in S. aureus). In E. coli, the activity of PhoBR is controlled by the PstSCAB phosphate transporter and the PhoU accessory protein. Loss of either PstSCAB or PhoU in E. coli and other bacteria results in constitutive activation of PhoBR. Preliminary investigations have revealed that S. aureus possesses three phosphate transporters, PstSCAB, PitA, and NptA, that expand the environments where S. aureus can obtain phosphate and contribute to infection. In S. aureus, both PstSCAB and NptA, but not PitA, are dependent on PhoPR for expression. Further differing from E. coli, S. aureus possesses three PhoU homologs, one associated with each transporter. Surprisingly, loss of either PstSCAB or the canonical PhoU homolog does not result in constitutive activation of PhoPR in S. aureus. These and other observations demonstrate that phosphate homeostasis in S. aureus differs from established models. Loss of PhoPR or PstSCAB and NptA reduces the ability of S. aureus to cause infection. Intriguingly, the ?phoPR and ?pstSCAB?nptA mutants do not phenocopy each other. The ?phoPR mutant has a reduced ability to grow in phosphate-limited environments and cause infection relative to the transporter double mutant. These observations indicate that PhoPR regulates additional unknown transporter-independent factors that enable S. aureus to survive phosphate limitation and cause disease. Together, these observations lead to the hypothesis that an expanded repertoire of regulatory proteins controls phosphate homeostasis in S. aureus and that disruption of this regulatory network reduces staphylococcal virulence. The two Aims of this proposal will test this hypothesis and elucidate the factors controlled by PhoPR that enable S. aureus to cause infection. Aim I will determine the contribution of PhoPR to S. aureus phosphate homeostasis and virulence. Aim II will evaluate the contribution of the three PhoU homologs possessed by S. aureus to controlling phosphate homeostasis.